1. Field of the Invention
The present invention relates to novel cell lines for use as a parent in hybridoma preparation which cell lines are established from human tumor cells other than those derived from bone marrow cells. The invention also relates to a method for establishing these novel cell lines, as well as novel hybridomas prepared by fusing cells of these parent cell lines with cells of another parent (hereunder referred to as partner) from human and animal cells. The invention further relates to a process for producing useful substance such as antibodies or lymphokines by culturing the novel hybridomas.
2. Description of the Prior Art
In the fields of immunology, biology, medical science and pharmacology, intensive efforts are being made to use as diagnostic agents or therapeutic agents the homogeneous and very highly specific antibodies (monoclonal antibodies) produced from hybridoma cells that can be grown in vitro and have the ability to produce antibodies to specific antigens. The possibility of preparing the hybridomas and using them in the production of biological substances in vitro was first demonstrated in 1975 by Ceaser Milstein and his co-workers at Cambridge University (Milstein C. et al, "Nature", 256, 495). They prepared a mutant cell line (P3-X63-Ag8) from a mouse myeloma strain (P3K) endowed by Leo Sacks of Salk Institute. The mutant cells were then fused with mouse spleen cells immunized with sheep red blood cells (SRBC). The resulting hybridoma was found to be capable of growing in vitro and producing monoclonal antibodies (MoAb) to SRBC. One great advantage of using hybridomas is that it provides a tool for mass production of biologically active substances by fusing tumor cells having high proliferative ability with those which can produce such biologically active substances but which usually have little or no proliferative ability in vitro.
Following the report by Milstein et al., many researchers made studies on hybridomas producing MoAb to specific antigens. In all of these studies, parent cells for use in the preparation of hybridomas were tumor cells derived from bone marrow cells such as myeloma cells and tumor cells from B cells. The genetic properties of tumor cells such as myeloma cells that are necessary for preparing hybridomas and the theoretical aspect of the hybridoma preparation are described hereunder in detail.
The first requirement for the preparation of a hybridoma from two or more kinds of parent cells is to use a system in which only the hybridized cells survive, the survival of the parents being prevented. Therefore, in most of the attempts at preparing hybridomas, tumor cells that can grow vigorously in vitro are such mutant cells as are deficient in either hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK). The genetic and biochemical behavior of both mutants is essentially the same, so the following description will be made based on the more common HGPRT-deficient cells. As is well known, HGPRT is one of the enzymes that is responsible in all cells for DNA synthesis by a salvage circuit in the route of the DNA synthesis. More specifically, if the DNA synthesis in the de novo circuit requiring components of purine or pyrimidine as substrates for the enzymatic reaction is suppressed by a certain inhibitor (e.g. aminopterin), the HGPRT actuates the salvage circuit as a rescue pathway and enables the continued DNA synthesis to keep the cells alive. Therefore, no HGPRT-deficient cells can survive in a medium containing hypoxanthine, aminopterin and thymidine (HAT medium) due to the presence of aminopterin which is an inhibitor against the de novo circuit. The partner cells for the preparation of a hybridoma, for example spleen cells (B cells), are capable of synthesizing DNA through both the de nove and salvage circuits. Therefore, the hybridoma produced by fusing the spleen cells with the HGPRT-deficient parent cells survives the inhibition of the de novo circuit by aminopterin in the HAT medium and effectively makes use of hypoxanthine to synthesize DNA through the salvage circuit originating from the spleen cells. In other words, the hybridoma posseses both the ability of the parent myeloma cells to grow vigorously in vitro and the ability to synthesize DNA by the salvage circuit originated from the spleen cells even under the inhibition of the de novo circuit in the presense of aminopterine in the HAT medium. Furthermore, the hybridoma has the genetic information from the spleen cells to produce immunoglobulins (antibodies) to specific antigens, so it is capable of growing in the HAT medium to produce immunoglobulins.
The HGPRT-deficient strain as a parent for the hybridoma preparation is usually selected as a cell line that is generated by a suitable mutation technique and which is resistant to 8-azaguanine and is unable to grow in the HAT medium. Conventionally, this parent consists of bone marrow derived tumor cells such as myeloma cells or B cell lymphoma. However, most of the tumor cells that can be cloned for use in the hybridoma preparation originate from mice or rats and human myeloma cells are seldom used. The reasons are: 1) clones that can be subjected to selection in the HAT medium (hereunder sometimes referred to as HAT selection) are difficult to prepare from human myeloma cells; 2) human myeloma cells do not have the high proliferative ability that is required for a parent, so a hybridoma prepared by fusing them with partner cells has only a limited ability to grow, and furthermore, it is not stable enough to produce a large amount of the desired substance such as immunoglobulins. Therefore, it has ben impossible in the art to prepare human-human hybridomas that have sufficient growing ability and produce useful substances. Researchers are unable to produce human immunoglobulins in quantity from cultured cells and instead they have to use immunoglobulins originating from animal-animal hybridoma cells. However, the immunoglobulins produced from animal-animal hybridomas are proteins which are foreign to humans, so their antigenicity prevents their extensive use in humans. Therefore, it is strongly desired to produce immunoglobulins from human-human hybridomas. But, as already mentioned, the mass cultivation of hybridomas from human myeloma cells involves great difficulty because of their instability.
Myeloma cells produce large quantities of immunoglobulins called myeloma proteins. However, it is not known whether the thus produced immunogloblins are antibodies to any specific antigens, and it is almost impossible to select myeloma cells producing an antibody to a specific antigen. Scientists have concluded that the presence of myeloma proteins complicates the screening of hybridomas or the purification of antibodies, so myeloma cells that produce myeloma proteins are being replaced by those which do not produce myeloma proteins. In other words, the growing ability in vitro is now regarded as a more important factor for the myeloma cells as a parent for the hybridoma preparation.
Under these circumstances, it is desired to obtain human cells as a parent cell line for hybridoma preparation that will grow vigorously and can be subjected to HAT selection after fusion with partner cells, as well as human hybridomas that remain sufficiently stable in vitro to produce large quantities of the desired substances such as immunoglobulins. It has been generally understood that the parent cells to be fused with partner cells for hybridoma preparation should be selected from among cancer cells derived from myeloma and B cells [including cells transformed by Epstein Barr virus (EBV)]. However, as mentioned before, these parent cells have various defects that prevent their extensive use in clinical applications.